Technical Field

The present disclosure relates to a pharmaceutical composition capable of inhibiting progression or development of cancer cells in a subject. The disclosed composition comprises at least a segment of peptide nucleic acid (PNA) capable of hybridizing onto a target protein or a complementary genetic sequence resided within the cancer cells to effectuate inhibition of cancer development. Furthermore, the present disclosure also pertains to a method to apply the disclosed composition in a predetermined manner to result the desired effect of prohibiting cancer development in the subject.

Background

Cancer stem cells (CSCs) are the major reason for the devastating effects of cancer (1). CSCs are critical factors that drive cancer aggressiveness, metastasis and drug resistance (1, 2). Therefore CSC-targeted cancer therapy is crucial for attaining the desired therapeutic outcome. However, finding a novel therapeutic agent capable of effectively eradicating the CSC cell population, despite being an ideal solution, has been proven extremely challenging in realization. Particularly, intratumour heterogeneous gene expression among cancer and CSCs are known to be a reason leading to failure of current targeted therapy in cancer as dysregulation of the expressed proteins appear to interrupt drugs that designed for aiming towards one or more target genes or their respective derivatives. Some studies even showed that CSCs can acquire resistance to EGFR (Epidermal Growth Factor Receptor) inhibitors, which is an effective therapy for lung adenocarcinoma patients with activating mutations, making effective therapy more difficult (4).

Nonetheless, advances in genomic technologies allows exploration of new avenue to yield new therapeutic agent effective in at least curbing progress or spreading of cancer cells in human subjects. For instance, PNA - a synthetic molecule substantially corresponds to the structure of DNA or RNA in cells - was found to possess greater affinity and stability for hybridizing on complementary nucleotide sequence thus rendering PNA a potential tool for cancer diagnostic, cancer treatment, or even as kit usable for DNA or RNA purification. In Chinese patent application no. 201610393487.3, a kit for detecting mutation of EGFR gene by hybridizing on the mutated region using PNA has been disclosed. Further, Terry et al. offer a modified PNA or PNA-conjugate for the treatment of glucocorticoid receptor betarelated disease in United States patent no. 10457947 by binding the glucocorticoid receptor gene using the modified PNA. Also, Korean patent application no. 20190023081 describes the implementation of PNA-based probe complementary to mutations found in genes relating to lung cancer progression to subsequently arrive at a diagnostic conclusion with respect to lung cancer in a human subject.

Further in view of the fact that long mononucleotide A-T repeats are cis-regulatory transcriptional controls of many genes, and the element increases the expression of many genes in various cancers, including lung cancer (5, 6). A-T repeats are cis-regulatory elements that regulate gene expression by binding onto Argonaute proteins (Agos) (5). Inventors of the present disclosure believe that disruption of A-T repeat interactions with Agos would dysregulate genes driving cancer development and potentially activate transcription of other cellular components beneficial to cancer patient in several ways. Thus, the binding of A and T repeats could be a targeted therapy or secondary treatment for cancer therapy. However, it is crucial to ascertain robustness and stability of the administered polynucleotide sequence complementary to A-T repeat for interrupting binding of Agos protein within the cancer cells resisting against various actions carried out by the nuclease present in the cells while exhibiting great affinity towards the target sequence to form a duplex or even triplex construct thereof to prohibit binding of Agos protein. As such, a synthetic PNA with specially designed and arranged nucleosides sequence may seem to be an ideal candidate to arrest, prohibit or cease cancer progress through downregulating expression of cancer promoting genes by disrupting binding of Agos protein.

Summary

One object of the present disclosure is to disclose a composition to arrest, stop, inhibit or incapacitate progress and development of cancer cells in a subject upon administering the disclosed composition to the subject.

Further object of the present disclosure aims to provide a composition effective against growth of cancer stem cells, which are resistant towards many cancer treatments, thus prohibiting further development of cancer in a human subject.

Still, another object of the present disclosure is directed to a method of prohibiting cancer progress or development in a subject using the abovementioned composition. The disclosed method capitalizes on excellent affinity and stability of the mentioned composition to competitively bind onto at least Argonaute proteins in the cancer cells and prevent expression of caner-promoting genes thereof thus arresting further development or spreading of cancer.

More object of the present disclosure relates to the use of the composition comprising synthetic counterpart of genomic A-repeats for preventing cancer progress and consequently eradicate the cancer cells off the subject.

At lease one of the above objects is met or partially met by one embodiment of the present disclosure pertaining to a composition for prohibiting development of cancer cells in a subject comprising a peptide nucleic acid oligomer comprising a first segment having a sequence of nucleobases consisting of a plurality of adenine; a carrier coupled to the oligomer to bring the oligomer into the cancer cells, wherein the oligomer is configured to compete with cellular RNA and/or DNA binding onto Argonaute proteins to downregulate expression of at least a gene capable of promoting development of the cancer cells.

In several embodiments, the oligomer has a total dimer number within the range of 10 to 30 to effectively work against the cancer progression. Preferably, the total dimer is in the range of 15 to 20 for competitively binding onto the Argonaute protein to attain the object to stop cancer advancement in the subject.

In more embodiments, the plurality of adenine is in a total number of 6-20. More preferably, the PNA contains a repeated adenine sequence of around 15 to 18 adenines.

For more embodiments, the genes being altered transcription are preferably those containing A- and/or T-repeats. In some embodiments, the gene being dysregulated or downregulated of its expression is any one or combination of BRWD1, FLOT2, SKA3, and TGF-pl. Expression of these gene are known to promote advancement of cancer stage while dysregulating their expression results potentially better reaction towards existing primary cancer treatment, reversal or slowing of cancer progression.

According to several embodiments, the oligomer is attached with one or more conjugate. The conjugate may serve to equip the oligomer with a desired property to attain better or almost better therapeutic outcome. For few embodiments, the conjugate is 8-amino-3-6- dioxaoctanoic acid, where presence of which may facilitate transportation of the oligomer into the cell or cell nucleus for manipulation of gene expression thereof.

Another aspect of the present disclosure refers to a method for inhibiting progress of cancer cells in a subject comprising administering therapeutically effective amount of a composition comprising a peptide nucleic acid oligomer comprising a first segment having a sequence of nucleobases consisting of a plurality of adenine; and bringing the oligomer into cancer cell such that the oligomer is configured to compete with cellular RNA and/or DNA binding onto Argonaute proteins to downregulate expression of at least a gene capable of promoting development of the cancer cells. Preferably, the oligomer has a total dimer number within the range of 10 to 30 and the plurality of adenine is in a total number of 6-20.

Another aspect of the present disclosure is directed to a peptide nucleic acid oligomer comprising a first segment having a sequence of nucleobases consisting of a plurality of adenine for use in a treatment of cancer. In a number of embodiments, the plurality of adenine is in a total number of 6-20 while the oligomer has a total dimer number within the range of 10 to 30

According to more embodiments, the disclosed oligomer further comprises a carrier and/or a conjugate attached to the oligomer. The carrier is 8-amino-3-6-dioxaoctanoic acid

Brief Description of Drawings

Fig. 1 shows the results about the expression of down- and upregulated genes containing A and T repeats where (a) exhibits a Western blot gel picture and corresponding graph for lysates of parental cells and CSC-enriched cells prepared for CD 133, CD44, ABCG2 and ALDH1 Al expression, (b) and (c) are graphs respectively depicting gene expression profiling with A and T repeats (X-axis) plotted against the repeat density in bp/Mbp (Y-axis) in spheroid and control samples for H292 and A549 cell lines (only perfect A and T repeats were classified as downregulated (Dn), neutral (Nu), or upregulated (Up) gene), (d) is a Venn diagram showing overlap between the intersection of downregulated genes between H292 (the left circle) and A549 (the right circle) cell lines, and (e) is a list of candidate genes determined by biological process from Gene Ontology (Student's t-test P-values are denoted by P indicating that the mean difference between Dn/Up and Nu is statistically significant and all plots present the mean ± SD (n = 3). * P < 0.05 vs. untreated cells). Fig.2 are graphs showing results of cytotoxicity tests against various cell lines where (a) H460, (b) H292, (c) H23, (d) A549, (e) HK2, (f) HEK293, and (g) human fibroblast cells were treated with various concentrations of PNA-A15 (0 - 40 pM) for 48 h or scramble, which was used as a control group with the cell viability being measure by MTT assay relative to the viability of untreated cells set as 100% and all plots represent the mean ± SD (n = 4). * P < 0.05 vs. untreated cells.

Fig. 3 shows results about effect of PNA-A15 in dysregulating tumour oncogene genes and tumour suppressor genes in lung CSCs where the dysregulated genes in (a) H292 and (b) A549 cancer cell lines were significantly increased and reduced compared with neutral gene (Dn, Nu, and Up denote downregulated, neutral, and upregulated genes, respectively, in spheroid H292 and A549 cancer cell lines compared with the scramble control in their spheroid cancer cell lines) that the repeat density was measured in bp/Mbp at 13-27 bp length, (c) is a Venn diagram showing overlap between the intersection of downregulated genes in H292 (the left circle) and A549 (the right circle) cell lines and (d) is a corresponding table of downregulated candidate genes determined by biological process from Gene Ontology, (e) is Venn diagram showing overlap between the intersection of upregulated genes between H292 (the left circle) and A549 (the right circle) cell lines and (f) is a table of upregulated candidate genes determined by biological process from Gene Ontology (Student's t-test P- values, which are denoted by P, indicate that the mean difference between Dn/Up and Nu is statistically significant. All plots show the mean ± SD (n = 3). * P < 0.05 vs. untreated cells).

Fig. 4 shows photos and corresponding graph about effect of PNA-A15 in inhibiting the anchorage-independent growth of human lung cancer in (a) H460, (b) H292, (c) H23, and (d) A549 cells, which were treated with PNA-A15 or scramble (SC) 5 pM) for 48 h and anchorage-independent colony formation was assessed by microscopy (4x) after 14 days of the treatment to measure the relative colony number and size of PNA-A15 or scramble- treated cells comparing to those of untreated controls (all plots present the mean ± SD (n = 3). * P < 0.05 vs. untreated cells).

Fig. 5 shows photos and corresponding graph about effect of PNA-A15 in suppressing CSC- like behaviour of human lung cancer in (a) H460, (b) H292, (c) H23, and (d) A549 cells, which were treated with PNA-A15 or scramble (SC) 5 pM) for 48 hours then cells were grown in a 24-well ultralow attachment plate at a density of 2.5 x 10 ³ cells/well for 7 days to form primary spheroids followed by trypsinizing the primary spheroids as single cells and replating for 14 days in a 24-well ultralow attachment plate to form secondary spheroids, which their number and size were determined and imaged using phase-contrast microscopy (Olympus 1X51 with DP70) at 14 days after replating with the relative spheroid number and size of PNA-A15- or scramble-treated cells being compared to those of untreated controls (all plots present the mean ± SD (n = 3). * P < 0.05 vs. untreated cells).

Fig. 6 are graphs showing effect of PNA-A15 in inhibiting the expression of CSC markers in (a) and (e) H460, (b) and (f) H292, (c) and (g) H23, and (d) and (h) A549 cells, which were treated with PNA-A15 or scramble 5 pM for 48 h with CD 133 expression levels being assessed by flow cytometry in CSC-enriched cells using anti-CD133 monoclonal antibodies followed by an Alexa Fluor 488-labelled secondary antibody (data represent the mean ± SD (n = 3). * p < 0.05 versus untreated control).

Fig. 7 shows results of PNA-A15 used in suppressing the expression of CSC-like phenotypes where (a) is a graph illustrating outcome of cell viability assessed using a WST-1 assay and relative cell viabilities compared to those of untreated controls, (b) are images of secondary spheroids of H292 cells treated with PNA-A15 or scramble 5 pM for 7 days followed by capturing the photos using lOx microscopy on days 0, 3, and 7 of the treatment) that the apoptotic and necrotic cell death on day 7 was assessed by Hoechst 33342/PI co-staining and visualized using fluorescence microscopy (10 ^x ), (c) is Western blot gel pictures and corresponding graph about the expression levels of CD 133, CD44, ABCG2 and ALDH1A1 in parental H292 cells treated with PNA-A15 or scramble 5 pM for 48 h, and (d) is Western blot gel pictures and corresponding graph about the expression levels of CD 133, CD44, ABCG2 and ALDH1A1 in enriched CSC H292 cells treated with PNA-A15 or scrambled 5 pM for 48 h days (blots were re-probed with p-actin to confirm the equal loading of samples and data represent the mean ± SD (n = 3). * P < 0.05 versus untreated control).

Detailed Description

Hereinafter, the disclosure shall be described according to the preferred embodiments and by referring to the accompanying description and drawings. However, it is to be understood that referring the description to the preferred embodiments of the disclosure and to the drawings is merely to facilitate discussion of the various disclosed embodiments and it is envisioned that those skilled in the art may devise various modifications without departing from the scope of the appended claim. Unless specified otherwise, the terms "comprising" and "comprise" as used herein, and grammatical variants thereof, are intended to represent "open" or "inclusive" language such that they include recited elements but also permit inclusion of additional, un-recited elements. As used herein, the terms “approximately” or "about", in the context of concentrations of components, conditions, other measurement values, etc., means +/- 5% of the stated value, or +/- 4% of the stated value, or +/- 3% of the stated value, or +/- 2% of the stated value, or +/- 1% of the stated value, or +/- 0.5% of the stated value, or +/- 0% of the stated value.

The term "gene" as used herein may refer to a DNA sequence with functional significance. It can be a native nucleic acid sequence, or a recombinant nucleic acid sequences derived from natural source or synthetic construct. The term "gene" may also be used to refer to, for example and without limitation, a cDNA and/or an mRNA encoded by or derived from, directly or indirectly, genomic DNA sequence.

The term “Argonaute protein” or “Agos” shall refer to at least one of the subunits from the Argonaute protein family contains one or more active site to bind with disclosed PNA.

The term “peptide nucleic acid” or “PNA” are used herein interchangeably referring to oligomers, which are artificially synthesized polymer similar to DNA or RNA with backbone composed of repeating N-(2-aminoethyl)-glycine units linked by peptide bonds. The PNA described in the present disclosure may take different lengths and modification to yield the desirable outcome in the subject. Further, PNA-A15 is one of several embodiments of the PNA used in the present disclosure to better illustrate the effects and results of the present disclosure.

One aspect of the present disclosure refers to composition for prohibiting development of cancer cells in a subject comprising a peptide nucleic acid oligomer comprising a first segment having a sequence of nucleobases consisting of a plurality of adenine; a carrier coupled to the oligomer to bring the oligomer into the cancer cells. It is important to note that the oligomer is configured to compete with cellular RNA and/or DNA binding onto Argonaute proteins to downregulate expression of at least a gene capable of promoting development of the cancer cells. As shown in the examples provided hereinafter, inventors of the present disclosure found that the disclosed composition is effective against development of cancer cells in any one or combination of lung cancer, pancreas cancer, esophageal cancer, cervical cancer, breast cancer, and stomach cancer. For some embodiments, the oligomer has a total dimer number within the range of 10 to 35. The total length of the oligomer used may render the disclosed composition operating differently within the cancer cell to dysregulate the cancer promoting genes and/or cancer suppressing genes. In several embodiments, the oligomer may comprise a second segment incorporated to the first segment. The second segment is implemented to carry out tasks varied from the first segment but eventually being directed to hinder progress of the cancer cells in the subject upon administering the disclosed composition to the subject. For example, the second segment may have a sequence resulting to enhanced affinity, improved specificity, etc.

Pursuant to several embodiments, the plurality of adenine is in a total number of 6-20 to reach the desired outcome. Oligomer used in the disclosed composition with relatively longer or greater number of adenine may be able to block away binding of the housekeeping genes expression of which are critical for survival of the cancer cells. Specifically, the first segment comprises lengthier or greater number of adenine shall lead to more efficient competitive binding onto the Argonaute proteins. This may completely or almost completely shield the binding site on the Argonaute proteins for the A- or T- repeat portion on the genomic RNAs required to initiate the expression of the cancer promoting genes. On the other hand, shorter adenine sequence tends to only work against more towards tissue-specific genes with corresponding mRNA having shorter A- or T- repeats. Dysregulating expression of the tissue specific genes is believed to have less impact about hindering cancer development in the subject. It was found by inventors of the present disclosure that the oligomer comprising around 12 to 20 adenine in length is preferably used. More preferably, oligomer comprising a first segment with about 14 to 16 adenines is used for preparation of the disclosed pharmaceutical composition. Under certain circumstances, the first segment of the oligomer may directly hybridize onto the A- or T- repeats of the genomic DNA and/or RNA to prevent any potential interaction with the Argonaute proteins thus hindering expression of the cancer promoting genes.

In a number of the embodiments, the gene being dysregulated of its expression is any one or combination of those containing A- and/or T- repeats as described in one or more examples provided in the present disclosure. One skilled in the relevant field shall appreciate the fact that interfering regulation one or more of these genes may alternatively bring about altering regulation in expression of other genes, which serve to effectuate an outcome contradictory to cancer promoting gene being dysregulated. Literally, applying the disclosed composition in a predetermined fashion and dosage can dysregulate expression of cancer developing gene that can be beneficial to the subject undergoing a primary cancer treatment.

To facilitate delivery of the disclosed composition to the cancer cells, the disclosed composition comprises a carrier in a number of preferred embodiments. For some embodiments, the carrier can be in a form of chemical modification to improve uptake of the PNA by the cancer cells. For example, polyarginine (poly-R) tails can be incorporated to the PNA of the disclosed composition for better cellular delivery. Further embodiments may involve chemical modification to the PNA backbone to change the polarity thus enabling better delivery of the disclosed composition to the site requiring the desired outcome to halt the cancer development. Chemical entities capable of introducing cationic amino acid side chains to the PNA backbone is applicable for such modification. For instance, in some embodiments, the carrier is 8-amino-3-6-dioxaoctanoic acid coupled to the oligomer to favor translocation of the oligomer into the cancer cells. Other alternative carrier systems known in the field facilitating uptake of the PNA oligomer by eukaryotic cells such as liposomes, polymer nanoparticles, co-transfection with partially complementary DNA, etc. can be employed in the disclosed composition as long employment of these carrier system will not adversely affect the therapeutic outcome. Still, further embodiments of the disclosed composition can include a conjugate attached to the oligomer. The conjugate equips the oligomer with extra functionality other than enhanced delivery offered by the carrier. For example, the conjugate can be a signaling entity, a cleavable moiety, etc. to provide

Further aspect of the present disclosure involves a method for inhibiting progress of cancer cells in a subject comprising administering therapeutically effective amount of a composition comprising a peptide nucleic acid oligomer comprising a first segment having a sequence of nucleobases consisting of a plurality of adenine; and bringing the oligomer into cancer cell such that the oligomer is configured to compete with cellular RNA and/or DNA binding onto Argonaute proteins to downregulate expression of at least a gene capable of promoting development of the cancer cells.

As setting forth, the oligomer employed in the disclosed method has a total dimer number within the range of 10 to 35 to better dysregulate the cancer promoting genes and/or cancer suppressing genes. Likewise, in several embodiments, the oligomer may comprise a second segment incorporated to the first segment dedicated to performing addition function for hindering the advancement of the cancer cells or cancer stage in the subject. Furthermore, the plurality of adenine is in a total number of 6-20 to reach the desired outcome despite the total length of the oligomer may restrict the number of the plurality of adenine available. The present disclosure found that relatively lengthier adenine sequence is prone to block the binding site resided in the Argonaute proteins essential for expression of the cancer promoting genes completely or almost completely. Thus, the oligomer shields the binding site on the Argonaute proteins from reacting with the A- or T- repeat portion of the genomic RNAs that the expression of the cancer promoting genes cannot be initiated in the absence of interaction with the Argonaute proteins. Preferably, the oligomer comprising around 12 to 20 adenine in length is employed in the disclosed method. More preferably, oligomer comprising a first segment with about 14 to 16 adenines is administered to the subject to at least hinder advancement, progression or development of further cancer stage. Like mentioned in the foregoing, the first segment of the oligomer may directly hybridize onto the A- or T- repeats of the genomic DNA and/or RNA to prevent any potential interaction with the Argonaute proteins in several other embodiments of the disclosed method

Referring to some embodiments of the disclosed method, the gene being dysregulated of its expression is by way of A- and/or -T repeats flanking about the relevant genes and the respective derivatives interaction with the Argonaute proteins.

Still, another aspect of the present disclosure associates to a peptide nucleic acid oligomer comprising a first segment having a sequence of nucleobases consisting of a plurality of adenine for use in a treatment of cancer. The cancer treatable using the disclosed oligomer can be any one or combination of lung cancer, pancreas cancer, esophageal cancer, cervical cancer, breast cancer, and stomach cancer.

For some preferred embodiments, the plurality of adenine is in a total number of 6-20 while the oligomer has a total dimer number within the range of 10 to 30. Moreover, in some embodiments, the disclosed oligomer further comprises a carrier and/or conjugate to facilitate delivery of the disclosed oligomer to the cancer cells specifically. Preferably, the carrier is 8- amino-3-6-dioxaoctanoic acid in several embodiments.

More aspect of the present disclosure regards to the use of a peptide nucleic acid oligomer comprising a first segment having a sequence of nucleobases consisting of a plurality of adenine in downregulating expression of at least a gene capable of promoting development of the cancer cells in a subject.

The following example is intended to farther illustrate the disclosure, without any intent for the disclosure to be limited to the specific embodiments described therein.

Example 1

Human NSCLC-derived cell lines H460, H23, H292, and A549; human kidney cells HK2 and HEK293; and fibroblasts were obtained from the American Type Culture Collection (ATCC®, Manassas, VA, USA). H460, H23, and H292 cells were cultivated in Roswell Park Memorial Institute (RPMI) 1640 medium supplemented with 10% foetal bovine serum (FBS), 2 mM L- glutamine, 100 U/mL penicillin and 100 pg/mL streptomycin. A459, fibroblast, HK2, and HEK293 cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% FBS, 2 mM L-glutamine, 100 U/mL penicillin and 100 pg/mL streptomycin. Cells were incubated in a 37 °C humidified incubator with 5% CO2 and were routinely subcultured using a 0.25% trypsin solution with 0.53 mM EDTA. RPMI 1640 medium, DMEM medium, FBS, L-glutamine, penicillin/streptomycin, phosphate-buffered saline (PBS), trypsin and EDTA were obtained from GIBCO (Grand Island, NY). In addition, 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), DMSO, Hoechst 33342, propidium iodide (PI), and bovine serum albumin (BSA) were purchased from Sigma Chemical, Inc. (St. Louis, MO, USA). Antibodies directed against CD44, ABCG2, ALDH1A1 and [3-actin and the respective secondary antibodies were purchased from Cell Signaling (Danvers, MA, USA). Antibodies directed against CD 133 were purchased from Cell Applications, Inc. (San Diego, CA, USA). PNA-A15 was synthesized from PANAGENE (PANAGENE, Deajeon, Korea. Cells were treated with either PNA-A15 or scramble (control) for 48 h. The culture medium was replaced by medium containing PNA- A15 or scramble every day. The cells were then collected for microarray, anchorageindependent growth, spheroid formation, flow cytometry, and Western blotting analysis.

Cells were harvested by centrifugation, and resuspended cells were incubated with lysis buffer (20 mM Tris-HCl (pH 7.5), 1% (v/v) Triton X-I00, 150 mM sodium chloride, 10% (v/v) glycerol, 1 mM sodium orthovanadate, 50 mM sodium fluoride, protease inhibitor cocktail (Roche Molecular Biochemical) and 100 mM phenylmethylsulfonyl fluoride) for 30 min on ice. The cellular lysates were collected, and their protein contents were determined using a BCA protein assay kit (Pierce Biotechnology, Rockford, IL, USA). Equal amounts of protein from each sample (80 pg for the CSC marker in the total cell population or 30 pg for other proteins and enriched CSC experiments) were separated by SDS-PAGE and transferred to 0.45-pm nitrocellulose membranes (Bio-Rad). The resulting blots were blocked for 1 h with 5% (w/v) nonfat dry milk in TBST (25 mM Tris-HCl (pH 7.5), 125 mM NaCl and 0.1% (v/v) Tween 20) and incubated with the appropriate primary antibodies at 4 °C overnight. After three washes in TBST, the blots were incubated with horseradish peroxidase (HRP)- conjugated secondary antibodies for 2 h at room temperature. Finally, protein bands were detected using an enhanced chemiluminescence substrate (Supersignal West Pico; Pierce, Rockford, IL, USA) and quantified using the analyst densitometry software package (BioRad).

Example 2

The formation of spheroids was performed under serum-free conditions in an ultralow attachment plate as previously reported (7). NSCLC-derived H292 and A549 cells were pretreated with PNA-A15 and scramble (5 pM) for 48 h. Then, cells were detached using 1 mM EDTA and suspended into single cells. These cells were grown in a 24-weIl ultralow attachment plate at a density of 2.5 x 10 ³ cells/well in stem cell media (SCM) (i.e., 0.8% (w/v) methylcellulose-based serum-free medium (Stem Cell Technologies, Vancouver, BC, Canada) supplemented with 20 ng/ml epidermal growth factor (BD Biosciences, San Jose, CA, USA), 20 ng/ml basic fibroblast growth factor and 4 mg/ml insulin (Sigma) for 7 days to form primary spheroids. These primary spheroids were harvested, resuspended as single cells using 1 mM EDTA and cultured in SCM for 14 days in a 24-well ultralow attachment plate to form secondary spheroids.

Example 3

Total RNA was extracted using TRIzol (Invitrogen Life Technologies, Carlsbad, USA); purity and integrity were evaluated using the ND- 1000 Spectrophotometer (NanoDrop, Wilmington, USA) and Agilent 2100 Bioanalyzer (Agilent Technologies, Palo Alto, USA). Total RNA was amplified and purified using the TargetAmp-Nano Labelling Kit for Illumina Expression BeadChip (EPICENTRE, Madison, USA) to yield biotinylated cRNA according to the manufacturer’s instructions. After purification, cRNA was quantified using the ND- 1000 Spectrophotometer (NanoDrop, Wilmington, USA). Next, 750 ng of labelled cRNA samples were hybridized to each Human HT-12 v4.0 Expression Beadchip for 18 h at 58 °C according to the manufacturer's instructions (Illumina, Inc., San Diego, USA). Detection of the array signal was performed using Amershamfluorolink streptavidin-Cy3 (GE Healthcare Bio-Sciences, Little Chalfont, UK) following the bead array manual. Arrays were scanned with an Illumina bead array Reader confocal scanner according to the manufacturer's instructions. To investigate whether A and T repeats regulate altered gene expression in spheroid formation, two groups of spheroid H292 and A549 cells were compared with H292 and A549 parental cells. Furthermore, whether PNA-A15 alters the expression of genes containing A and T repeats was also assessed. Two groups of spheroids, H292 and A549 cells, were transfected with PNA-A15 and scramble. Each group consisting of 2 samples was studied. Data were uploaded to the Gene Expression Omnibus (GEO: GSE142616).

The down- and upregulated genes in microarray experiments were identified by CU-DREAM (P-value threshold = 0.05) (8). Microarray samples are shown in Table 1. The present disclosure intersected the down- and upregulated genes between the two experiments. Next, the present disclosure searched for A-T repeats around the transcription start sites (TSSs) of those genes (5). The human genome (GRCh38) was downloaded via Entrez Direct (EDirect) and its software package ncbi-entrez-direct. The chromosomal locations of genes were downloaded from ftp://ftp.ncbi.nlm.nih.gov/gene/DATA/gene2refseq.gz. The genome sequence around a TSS was divided into 200 bins, and each bin was 100 bp in length. Bins 1 to 100 covered 10,000 bp upstream of a gene, whereas bins 101 to 200 covered a 10,000-bp intragenic region. In each bin, intact A repeats (length 13-27 bp) were identified, and their lengths were summed. For instance, a bin consisted of three A repeats that were 12, 13, and 15 in length. The length sum of this bin was 12 + 13 + 15 = 40. A repeat that overlapped between two bins contributed to each bin proportionately.

CSCs from H292 and parental cell lines were prepared following previous studies (7, 10) and tested for CD 133, CD44, ABCG2, and ALDH1A1, which are well-known CSC markers (10, 11 ). Inventors of the present disclosure found that the expression levels of CSC markers CD 133, CD44, ABCG2 and ALDH1A1 were all dramatically increased in the enriched CSC population compared to their parental cells (Fig. la).

A previous study demonstrated that genes containing long mononucleotide A-T repeats are overexpressed in various cancers (6). To determine whether A-T repeats regulate genes differently between lung cancer and CSCs, the present disclosure compared A-T repeats around TSS to the spheroid formation population of H292 and A549 lung cancer cells. Inventors of the present disclosure divided the expression profiling of genes in the spheroid population compared to the total population of their parental cells into 3 groups, up, down, and neutral, depending on the levels of mRNA. The results showed that the T repeat densities of downregulated genes were significantly increased compared with those of neutral genes in the spheroid population of H292 and A549 cell lines (P-values = 1.18E-04 and 4.65E-10, respectively). These results suggest that genes containing A-T repeats of cancer cells and CSCs are differentially expressed (Fig. lb, c).

Then, the candidate genes were selected from overlapping T repeat densities of downregulated genes between H292 and A549 cells and were classified by a biological process based on Gene Ontology analysis as shown in Fig. Id and Supplementary Table SI. Fig. le shows that the 75 overlapping genes were filtered with a P-value < 0.05 in both H292 and A549 cells and were related to genes depending on their functions. The present disclosure found that dysregulated A and T repeat densities of CSCs in both cell lines were different.

Example 4

For the cytotoxicity assay, H460, H23, H292, A549, HK2, HEK293 and human fibroblasts were seeded onto 96-well plates at a density of 10 x 10 ⁴ cells/well and allowed to adhere by incubation overnight. Cells were then treated with various concentrations of PNA-A15 or scramble plasmid (0 - 40 pM) for 48 h and then analysed for cell viability using the 3-(4,5- dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Cells were incubated with 500 pg/ml MTT for 4 h at 37 °C, and the intensity of the formazan product after solubilization in 100 pl DMSO was measured at 570 nm using a microplate reader (Anthros, Durham, NC, USA). Relative cell viabilities were calculated by dividing the absorbance of the treated cells by that of the control cells. The half-maximal inhibition concentration (ICso) was determined from four independent experiments using GraphPad Prism 5.0 software (La Jolla, CA).

Prior to determining the effect of PNA-A15 on CSC properties, the appropriate noncytotoxic concentrations were evaluated. To study the effect of PNA-A15, human NSCLC H460, H292, H23, and A549 cells were treated with various concentrations of compound (0, 2.5, 5, 10, 20, and 40 pM) for 48 h, and then their cell viability was determined by the MTT viability assay. PNA-A15 was found to be nontoxic at concentrations below 5 pM, whereas no cytotoxic effect was found for the scramble control (Fig. 2a - d). Interestingly, PNA-A15 showed no cytotoxic effect on normal cells that contained HK2, HEK293 and human fibroblasts at all tested concentrations (0 - 40 jiM) (Fig. 2e - g). Thus, nontoxic concentrations of PNA-A15 were further tested for their effects on CSC-like gene expression and phenotypes.

Example 5

After treatment, cells were subjected to an anchorage-independent growth assay based on colony formation in soft agar. The bottom layer was prepared using a 1:1 (v/v) mixture of RPMI-1640 or DMEM medium containing 10% (v/v) FBS and 1% (w/v) agarose. This mixture was allowed to solidify for 30 min, after which the upper cellular layer composed of RPMI-1640 or DMEM medium with 0.3% (w/v) agarose, 10% (v/v) FBS and 1 x 10 ³ cells/ml was prepared and added. Finally, RPMI-1640 or DMEM medium containing 10% (v/v) FBS was added over the upper layer, and the cells were incubated at 37 °C for 14 days. Afterwards, colony formation was observed and imaged using phase-contrast microscopy (Olympus 1X51 with DP70). The relative colony numbers and sizes were calculated relative to those for the untreated cells.

The ability of cancer cells to form 3D spheroids and to grow in anchorage-independent conditions is widely used to assess CSC phenotypes (2, 10). Therefore, the present disclosure investigated the role of PNA-A15 in CSC behaviours. Accordingly, the effect of PNA-A15 on the growth and survival of NSCLC-derived cells was evaluated under these conditions. First, H460, H292, H23 and A549 cells were exposed to noncytotoxic concentrations of PNA-A15 for 48 h and subsequently analysed for colony formation in soft agar by recording the relative number and area of colonies after 14 days compared to untreated control or scramble-treated cells (Fig. 4). The present disclosure found that the PNA-A 15 -treated cells exhibited a significant decrease in the number and size of colonies compared to the untreated or scramble-treated control cells. Then, the spheroid formation in ultralow attachment plates was evaluated. The present disclosure found that treatment of the cells with PNA-A 15 significantly inhibited spheroid formation (Fig. 5). These results suggest that PNA-A 15 inhibits the growth and survival characteristics of CSCs in NSCLC-derived cells. To determine lung CSC markers, immunocytochemistry experiments assessing the expression of CD 133 were performed and analysed by flow cytometry. The results showed that PNA-A 15 treatment decreased the mean fluorescence intensity of CD 133 compared with the untreated or scramble-treated control (Fig. 6).

Example 6

Spheroid viability was determined using a water-soluble tetrazolium salt (WST) surrogate viability assay according to the manufacturer’s instructions (Roche Diagnostic GmbH, Mannheim, Germany). Briefly, at day 14 of secondary spheroid formation, as detailed in the “Single 3D spheroid formation assay” section, the cells were incubated with 10% (v/v) WST- 1 at 37 °C for 30 min. The intensity of the formazan product was determined using a microplate reader (Anthros, Durham, NC, USA), and the relative cell viability (%) was calculated as the absorbance of PNA-A15 or scramble-treated cells relative to untreated cells.

For CSC-rich population establishment, the spheroid culture assay used in this study was slightly modified from a previously described method (2, 9, 10). Cells were seeded onto a 24- well ultralow attachment plate with approximately 2.5 x 10 ³ cells/well in 0.8% methylcellulose-based serum-free medium supplemented with 20 ng/ml epidermal growth factor and 4 mg/ml insulin. The primary spheroids were allowed to form for 7 days. At day 7 of primary spheroid culture, primary spheroids were resuspended into single cells using 1 mM EDTA. Again, 2.5 x 10 ³ cells/well were seeded onto a 24-well ultralow attached plate. Secondary spheroids were allowed to form for 14 days. For the single three-dimensional (3D) spheroid-formation assay, cells were allowed to form primary and secondary spheroids as detailed above. At day 14 of secondary spheroid formation, they were dissociated into a single spheroid of the same size, and each spheroid was then treated with 5 pM PNA-A15 or scramble and allowed to grow in the indicated time. Phase-contrast images of the secondary spheroids were obtained at days 0, 3 and 7 after PNA-A15 or scramble treatment under a phase-contrast microscope (Nikon Eclipse Ts2).

Example 7

H460, H292, H23 and A549 cells were pretreated with PNA-A15 and scramble (5 pM) for 48 h, and then, cells were plated and allowed to form primary and secondary spheroids as detailed above. At day 14 of the secondary spheroid, spheroids were harvested by centrifugation and suspended into single cells using 1 mM EDTA. The resuspended cells were incubated on ice with a rabbit anti-CD133 antibody for 1 h. Next, the primary antibody was removed, and the cells were washed and incubated for 30 min with an Alexa Fluor 488- conjugated goat anti-rabbit IgG (H+L) secondary antibody (Life Technologies, Eugene, OR, USA). After washing, the fluorescence intensity was determined by flow cytometry using a 488-nm excitation beam and a 519-nm bandpass filter (FACSort; Becton Dickinson, Rutherford, NJ, USA). The mean fluorescence intensity was quantified using CellQuest software (Becton Dickinson).

At day 14 of secondary spheroid formation, these spheroids were dissociated into a single spheroid of the same size, and each spheroid was then treated with 5 pM PNA-A15 or scramble and allowed to grow in the indicated time. Phase-contrast images of the secondary spheroids were obtained at days 0, 3 and 7 after PNA-A15 or scramble treatment under a phase-contrast microscope. On day 7, every individual spheroid was incubated with 10 pg/ml Hoechst 33342 for 30 min followed by 5 pg/ml PI for 5 min. Nuclear condensation and DNA fragmentation of apoptotic cells were visualized using a fluorescence microscope (Olympus 1X5; 40x) equipped with a DP70 digital camera system (Olympus, Tokyo, Japan).

The present disclosure further monitored the effect of PNA-A15 on the maintenance of CSCs in the CSC-rich population. The spheroids were either treated with PNA-A15 or scramble plasmid or untreated for 48 h. Cell viability was then evaluated by WST assay. PNA-A15 significantly decreased the viability of CSCs in H292 cells compared to parental and scrambled cells (Fig. 7a). Every individual spheroid was subsequently treated with PNA-A15 at 5 pM for 0 - 7 days. It was found that PNA-A15 treatment significantly decreased the size of the spheroids and induced apoptosis at days 3 and 7 after treatment as determined by increased chromatin condensation and nuclear fluorescence following Hoechst 33342 and PI staining (Fig. 7b). These results support the above observed inhibitory effect of PNA-A15 on CSCs.

Furthermore, CSC markers, including CD 133, CD44, ABCG2 and ALDH1A1, were analysed by Western blot analysis. Fig. 7c shows that PNA-A15 caused a significant decrease in CD133, CD44, ABCG2 and ALDH1A1 expression compared with the untreated or scramble- treated control level in parental cells. The results were confirmed in the 3D-CSC-enriched population. The enriched CSC population was treated with PNA-A15 at 5 pM for 48 h, and the expression of CD 133, CD44, ABCG2 and ALDH 1A1 was determined by Western blot analysis. Fig. 7d shows that PNA-A15 treatment reduced CD 133, CD44, ABCG2 and ALDH1A1 expression compared with the untreated or scramble-treated control level. These results strongly support the role of PNA-A15 as a multiple-gene targeting agent for lung CSCs.

Example 8

The present disclosure determined genome-wide gene expression after spheroid cancer cell transfection with PNA-A15. Cells were pretreated with PNA-A15 or scramble PNA oligo for 2 days and then evaluated for spheroid formation in ultralow attachment plates. After that, pretreated cells were seeded at a low density and allowed to form primary spheroids for 7 days. The primary spheroids were then resuspended into single cells, and secondary spheroids were allowed to grow for 14 days. Then, inventors of the present disclosure analysed the expression profiling of A and T repeat genes in the CSC-rich population treated with PNA- A15 compared to their scramble cells. The present disclosure found that the A repeat densities of upregulated genes were significantly reduced compared with those of neutral genes )P-values = 1.30E-08 ,(and the T repeat densities of downregulated and upregulated genes were also significantly altered compared with neutral )P-values =3.69E-04 and 1.65E- 19, respectively (in H292 spheroids .Furthermore, T repeat densities of down -and upregulated genes were also significantly altered compared with neutral genes )P-values= 6.53E-07 and 2.39E-02, respectively (in spheroid A549 cells. These results suggest that PNA- A15 significantly alters the expression of genes containing A- and T- repeats in spheroid H292 and A549 cancer ceil lines (Fig. 3a, b).

To determine PNA-A15 dysregulated genes in CSCs, inventors selected candidate genes from overlapping A and T repeat densities of both down- and upregulated genes between the CSC- rich population treated with PNA-A15 and their scramble cells. All genes were classified by a biological process based on Gene Ontology analysis as shown in Fig. 3c and Supplementary Table S2. The validation genes were selected based on function and their biological processes. The data represent 69 overlapping genes filtered with a P-value < 0.01, and the present disclosure found that PNA-A 15 dysregulated the A and T repeat densities of downregulated genes that are related to genes acting as proto-oncogenes and inducing tumorigenesis in both cell lines. PNA-A 15 dysregulated the A- and T- repeat densities of upregulated genes, which act as a tumor suppressor and induce apoptotic processes (Fig. 3e, f and Supplementary Table S3).

Here, the present disclosure found that A and T repeats regulate gene expression in CSCs; therefore, PNA-A 15 represents a promising targeted therapy agent in lung cancer by downregulating multiple oncogenes and upregulating multiple tumor-suppressor genes. The present disclosure also revealed that A-T repeat-containing genes that are downregulated in CSCs are involved in CSC morphology, such as BRWD1, FLOT2, SKA3, and TGF-pi. BRWD1 is a transcriptional activator that inhibits cell proliferation by coordinately regulating MYC, and this mechanism plays a role in the self-renewal of CSCs (12). TGF- 1 is a multifunctional cytokine that plays a critical role in the regulation of CD 133 expression. Previous work revealed that TGF-01 upregulates CD 133 expression in hepatocellular carcinoma (13). FLOT2 is a proto-oncogene that regulates cell migration and invasion in cancer. FLOT2 is involved in cell cycle progression and regulates signaling pathways that are responsible for sternness and induce cadherin binding (14). FLOT2 is necessary for TGF-pi and induced epithelial-mesenchymal transition (EMT) in gastric cancer. Suppression of FLOT2 results in decreased cell invasion through repressing TGF-pi -mediated EMT and decreased CSCs (14, 15). Moreover, SKA3 is a proto-oncogene that induces cell cycle progression. Previous work revealed that SKA3 mRNA expression was elevated and correlated with poor survival outcomes in lung cancer patients (16). SKA3 acts as an oncogene by directly binding to EGFR and promoting cancer metastasis (16, 17). Moreover, the SKA3 gene acts as a proto-oncogene and induces tumorigenesis by inhibiting p53 in the apoptotic process. Additionally, p53 regulates the expression of CSC markers. Thus, activating p53 not only increases apoptotic induction in tumor cells but also suppresses CSC self-renewal.

After treatment of PNA-A15 into spheroids of H292 and A549 cells, the present disclosure found that the expression of many A and T repeat-containing genes changed in both the up- and downregulation directions. CSCs are difficult to therapeutically target because they are inherently resistant to cytotoxic and targeted drugs, and they also evade radiotherapy and immune surveillance (1, 18). Recent data show that CSC populations underlie significant diversification and plasticity and disguise heterogeneity (18). Our experiments also demonstrated heterogeneity in the gene expression of CSCs. This heterogeneity prevents the effectiveness of therapy targeting a single target. Therefore, PNA-A15 is a promising targeted therapy in lung cancer by downregulating multiple oncogenes and upregulating multiple tumour suppressor genes. PNA-A15 treatment downregulated A-T repeat-containing genes. Interestingly, many of the genes possess stem cell maintenance and functions, such as the ARID 1 A, MAPKI2, PLOD3, and RAC1 genes (19-22). Previous studies revealed that AR1D1 A regulates SOX9 expression, which is a stem cell transcription factor that maintains the self-renewal property of intestinal stem cells (19). In contrast, the deletion of the ARID 1 A gene results in the loss of stem cells and increases apoptosis in adult mice (19, 23). MAPK12 also modulates the sternness of various CSC types by inducing tumorigenesis and cancer aggressiveness (21). A previous study revealed that MAPK.12 significantly increased EMT and promoted CSC regulation in breast cancer (21). In addition, PNA-A15 interferes with both cancer and CSC phenotypes through the regulation of PLOD3, which is a potent inducer of lung cancer metastasis via the RAS-M APK pathway in vivo (22). Moreover, PLOD3 also interacts with STAT3, which is a stem cell transcription factor that maintains the self-renewal property of lung stem cells (22). PNA-A15 also downregulated the RAC1 gene, which mediated lung tumour growth and increased cell proliferation in vivo. Previous work demonstrated that RAC1 is critically involved in NSCLC migration and lung CSC formation and that RAC1 served as a useful therapeutic target by inhibiting tumour initiation and metastasis of CSCs in lung cancer (20). Thus, PNA-A15 not only interferes with tumour cells but also suppresses several genes possessing the self-renewal of CSC functions.

PNA-A15 treatment upregulated A-T repeat-containing genes that act as tumour suppressor genes, such as BAHD1, CCAR1, EGR1, and SLC5A11. These genes prevent tumour formation and increase apoptotic processes (24-26). CCAR1 is a tumour suppressor gene related to p53-induced apoptosis (27). BAHD1 is a tumour suppressor gene, and this antiproliferative protein inhibits cell cycle progression from G0/G1 to s phase (24). EGR1 is a tumour suppressor gene that regulates cell survival by activating the expression of p53 to prevent tumour formation (26). This role is important because p53 regulates the expression of CSC genes. Thus, activating p53 not only increases apoptotic induction in tumour cells but also suppresses self-renewal of CSCs. CCAR1 mediates the expression of multiple cell cycle regulatory genes and plays a role in cell cycle progression as a transducer of Notch signalling [26]. The Notch signalling pathway is involved in the process of stem cell initiation (28, 29). In some solid tumours, dysregulation of the Notch signalling pathway is correlated with tumour initiation and increased tumour sphere formation (30). These findings suggest that PNA-A15 may target the Notch signalling pathway in lung CSCs.

PNA-A15 is not toxic to normal cells but effectively kills cancer cells. Previously, the reported that short A and T repeats (2-9 bp) are more abundant in tissue-specific genes, whereas long A and T repeats (10-30 bp) are more abundant in housekeeping genes [5]. For normal cells, genes controlling differentiation are essential [5, 6]. On the other hand, to maintain the cancer and stem cell phenotypes, cancer cells must prevent foil differentiation and use housekeeping genes to play a major role in cell survival. Therefore, long A and T repeats may influence cancer and stem cell survival more than completely differentiated cells. In conclusion, A-T repeat transcriptional control is essential for CSC biology. Furthermore, interfering with PNA-A15 activity can disrupt CSC growth by dysregulating many genes.

This property of PNA-A15 suggests the potential of PNA-A15 in lung CSC treatment to overcome genetic heterogeneity. Moreover, PNA-A15 might be a potential therapeutic drug for many other CSCs in addition to lung cancer. It is to be understood that the present disclosure may be embodied in other specific forms and is not limited to the sole embodiment described above. However, modification and equivalents of the disclosed concepts such as those which readily occur to one skilled in the art are intended to be included within the scope of the claims which are appended thereto

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